The invention relates to novel chemokine-like proteins and their use to suppress the proliferation of actively dividing myeloid cells, e.g., myeloid progenitor cells, myeloid stem cells, and leukemic cells.
Each year approximately 173,000 of the people who undergo chemotherapy become neutropenic, which causes them to become susceptible to infection and anemia (Hecht, Drug and Market Development, 4:49, 1993). One method of treatment for neutropenia, e.g., chemotherapy-induced neutropenia, includes stimulation of progenitor cells with differentiation factors including granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), and erythropoietin (EPO) as a method of salvaging cells surviving chemotherapy. Approximately 100,000 patients annually are suitable for receiving G-CSF for this purpose. G-CSF is used to stimulate growth of white blood cell progenitors, and EPO has been used to stimulate production of red blood cells, however, there are no megakaryocyte progenitor stimulating factors currently available.
An alternative approach for preventing neutropenia is to inhibit cell proliferation with low doses of various chemokines, which inhibits cell cycling, thereby protecting the progenitor cells from the effects of chemotherapy and/or radiation therapy. After chemotherapy has ended, the chemokine treatment is also stopped, which allows the progenitor cells to resume normal proliferation.
Chemokines are small inducible proteins that are related by amino acid homology, chromosome location, and structural similarities, including the presence of four position-invariant cysteine residues in their primary amino acid sequence that form two disulfide bonds. The amino acid sequences of various naturally occurring, wild-type chemokine proteins are shown in FIG. 1.
Certain chemokines, known as beta chemokines, have a Cys--Cys pair as the first two cysteines, and include macrophage inflammatory protein-1 alpha (MIP-1.alpha.), MIP-1.beta., macrophage chemotactic and activating factor (MCAF, also known as monocyte chemo-attractant protein-1 (MCP-1)), MCP-3, and Regulated on Activation, Normal T-cell Expressed and Secreted protein (RANTES). The beta chemokines are potent chemoattractants for a variety of blood cell components, including monocytes, eosinophils, and T-lymphocytes, but not neutrophils.
Other chemokines, known as alpha chemokines, have a Cys-X-Cys triplet as the first two cysteines (X can be any amino acid other than cysteine), and include the human-derived proteins interleukin-8 (IL-8), GRO-.alpha. (also called melanoma-growth stimulating activity (MGSA/GRO), MIP-2.alpha. (also known as GRO-.beta.), MIP-2.beta. (also known as GRO-.gamma.), neutrophil activating peptide-2 (NAP-2), platelet factor 4 (PF4), gamma interferon inducible protein 10 (.gamma.IP-10), Epithelial derived Neutrophil Activating protein (78 amino acids in length)(ENA-78), .beta.-thromboglobulin (.beta.TG), connective tissue-activating peptide-III (CTAP-III), and platelet basic protein (PBP). The alpha chemokines are potent chemoattractants and all except PF4 and .gamma.IP-10 activate neutrophils.
Chemokines have been shown to regulate proliferation and/or differentiation of hematopoietic stem and progenitor cells in vitro and in vivo. For example, MIP-1.alpha., .gamma.IP-10, IL-8, Gro-.beta., PF4, and MCP-1 have been shown to inhibit the proliferation of colony forming unit-granulocyte macrophage (CFU-GM), burst forming unit-erythroid cells (BRU-E), and colony forming unit-multipotential progenitor cells (CFU-GEMM), at concentrations greater than 25 ng/ml when administered to mice. See, e.g., Broxmeyer et al., J. Immunol., 150:3448-3458 (1993).
On the other hand, several members of the chemokine family, including NAP-2, Gro-.alpha., Gro-.gamma., RANTES, and MIP-1.beta. have been shown not to possess such inhibitory activities, but Gro-.alpha. and Gro-.gamma. have been shown to interfere with the inhibitory activity of IL-8 and PF4. Furthermore, Broxmeyer et al., WO 94/13321, states that combinations of any two of MCAF, MIP-1.alpha., MIP-2.alpha., IL-8, .gamma.IP-10 and PF4 provide a decrease in the concentrations required for suppressing progenitor cell proliferation.
Several groups have also examined the ability of chemokines to inhibit the proliferation of progenitor cells in vivo. For example, Maze et al., J. Immunol., 149:1004 (1992), observed that murine MIP-1.alpha. suppressed proliferation and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells from mice femurs and spleens at doses between 2 and 10 .mu.g per mouse, injected intravenously. Murine MIP-1.beta., which was unable to inhibit cell proliferation in vitro, displayed no biological activity in vivo either.
Recombinant human chemokines have also been demonstrated to inhibit proliferation of CFU-GM, CFU-GEMM, and BFU-E following intravenous injection into mice. Furthermore, active chemokines cause a significant decrease in the number of progenitor cells in S-phase. For example, Dunlop et al., Blood, 79:2221 (1992), observed that recombinant human MIP-1.alpha. was able to suppress CFU-S in a dose dependent manner in vitro, and to reduce the high proliferative state of the CFU-S compartment to a quiescent state in vivo.
Human MIP-1.alpha. has been shown to protect progenitor cells in vivo from the cytotoxic effects of the chemotherapeutic drug cytosine arabinoside (ARA-C), and Caen et al., Blood, 82:162a (1993), has reported that PF4, at doses between 1 and 5 .mu.g/mouse, protected hematopoietic precursor cells from the adverse effects of the chemotherapeutic, 5-fluorouracil. Lord et al., Blood, 79:2605-2609 (1992), also observed that MIP-1.alpha. protected myeloid progenitors in a murine system from the cytotoxic effects of hydroxyurea.
In addition, Gewirtz et al., J. Clin. Invest., 68:56 (1989), observed that a peptide from PF4 containing the C-terminal 24 amino acids inhibited proliferation of megakaryocyte progenitors in vitro at a concentration of 25 .mu./ml. A shorter peptide containing only the last 13 amino acids from PF4 was found to be inactive in the assay. Similarly, a peptide containing the C-terminal 18 amino acids from .beta.-thromboglobulin did not inhibit proliferation.
Recently, Caen et al., Blood, 82:162a (1993), reported that a dodecapeptide, Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Glu-Ala-Pro, which is able to inhibit human and murine megakaryocyte and platelet production, can also protect hematopoietic precursor cells in vivo (1-5 .mu.g/mouse) during 5-fluorouracil chemotherapy.
Although G-CSFs are the drugs most commonly used to treat chemically-induced neutropenia, they have certain drawbacks. For example, G-CSFs do not prevent a drop in white blood cell count, i.e., they do not avoid neutropenia, but merely shorten the low point or nadir in the blood count. G-CSFs also fail to stimulate platelet development, and thus do not protect platelets. Moreover, G-CSFs are expensive, and are therefore often administered only after the white cell count drops below about 1000.